This proposal aims to differentiate stem cells for repair of diseased and damaged tissues and to evaluate tissue interactions in three-dimensional biomaterial mimics of the osteochondral disease microenvironment. The osteochondral microenvironment involves a complex milieu of multifaceted interactions between chondrocytes, osteoblasts, and mesenchymal stem cells (MSCs). In disease states, such as osteoarthritis and osteoporosis, this signaling is disrupted. Current repair strategies are limited by inadequate understanding of cell-cell communication, tissue interactions, and the impact of disease state and differentiation state on repair. These issues are compounded by traditional two-dimensional culture which is generally used to characterize cells and fails to accurately represent the native three-dimensional tissue architecture. Furthermore, traditional studies of cell interactions using conditioned media and transwell culture are limited by lack of proximity between cell types, thus reducing the concentration of soluble signaling factors and crosstalk between cells that may normally occur in vivo. Finally, controversy remains over whether the primary role of MSCs In repair is as a cell source or stimulator of other cellular repair. This proposal aims to evaluate the effects of cellular differentiation state, disease state, and the dual roles of MSCs in repair. This research will utilize bilayered poly(ethylene glycol) gels in addition to a novel co- culture system developed to simultaneously support cartilage and bone tissue formation. This system will consist of a layer of poly(ethylene glycol) to support cartilage tissue formation and a poly(lactic-co-glycolic acid) with hydroxyapatite layer to support bone tissue formation. Use of both systems will allow extensive analysis of the ability of MSCs to form tissue and stimulate repair of healthy and diseased tissues. It is hypothesized that intermediate stages of MSC differentiation will enhance MSC stimulation and formation of tissues. This project has the potential to impact delivery strategies for stem cells. Specifically, it may impact treatments of osteochondral ailments including osteoarthritis, osteoporosis, and other diseases of bone and cartilage. As the American population continues to age, the incidence of diseases involving both cartilage and bone (such as osteoarthritis and osteoporosis) continues to increase. This research seeks to enhance repair strategies in osteochondral diseases through increased understanding of cell and tissue interactions.